Receptor recognition and binding of ligands are remarkable events by which the numerous interactions are being controlled in the body. Especially, carbohydrates have a central role in biological phenomena such as protein carbohydrate interactions. Despite known role of carbohydrates in biology, relatively few investigations are reported on methods of enhancing these interactions. New targets for carbohydrate such as enzymes, proteins and viruses are being identified which can have numerous applications in therapeutics. Carbohydrates play critical role in various biological processes such as cell recognition, cell adhesion, cell differentiation, inflammation, viral and bacterial infection, tumerigenesis, and metastasis (Rouhi, A. M., Chem. Engg. News, Sep. 23, 62–66, 1996).
Sharon et al., (Science 246:227–234, 1989) reported that the carbohydrate portions-of glyco-conjugate molecules to be an important entity in carbohydrate biology. Advantage of carbohydrate modification lies in that it may impart change in physical characteristics such as solubility, stability, activity, antibody recognition and susceptibility to enzyme.
Carbohydrates can be utilized as binding entity to the receptors by incorporating in the monomer. Thereby the polymerizable monomers containing ligand can be oligomerized or polymerized to form a multivalent conjugate. Multivalent ligand may include shorter oligomers having pendant functional groups that may impart specific properties to the polymer.
A recent patent granted to Krepinsky, et al. (U.S. Pat. No. 6,184,368, 2001) suggests the application of carbohydrates in preventing the infections. Mandeville, et al. (U.S. Pat. Nos. 5,891,862, 1999 and 6,187,762, 2001) reported the use of polyvalent polymers containing carbohydrates for the treatment of rotavirus infection. Monovalent ligands display weak affinities and poor specificity towards the receptor binding sites. In contrast, a saccharide in a multivalent form can bind to the same substrate with greater affinity and specificity. The binding of cell surface receptors to multivalent carbohydrate molecules exhibits wide variety of biological responses and has unique edge over monovalent interactions (Mammen, et al., Angew. Chem., IntEd., 37, 2754–2794, 1998).
Multivalent ligands of varying length and density are useful for receptor ligand interactions in biological systems. Many chemical and chemoenzymatic methods have been reported for the preparation of di- and trivalent ligands, dendrimers, and high molecular weight polymers, but involve complex synthetic methods. Thus, there is a need to devise simple methodology to obtain multivalent ligands of varying polymolecularity.
Polyvalent ligands present on pathogen bind to multiple receptors on the host cell. Oligomers or polymers comprising multiple ligands could be more effective inhibitors for the host cell receptor, as a result of higher affinity for the pathogen. In addition the higher molecular weight of the polymeric ligands also prevents the infection through steric exclusion. (Spaltenstein, A., and Whitesides, G. M., J. Aru. Chem. Soc., 113, 686, 687, 1991).
Agglutination of erythrocytes caused by influenza virus can be prevented by use of polyvalent sialic acid inhibitors. This novel approach which is a model for pathogen host interactions was reported by Mammen, M., and Whitesides, G., M., (J. Med. Chem. 38(21), 4179–90, 1995). The authors reported polymers containing sialic acid as effective inhibitors of influenza virus. Moreover, they suggested two favorable mechanisms for inhibition between the surfaces of virus and erythrocytes 1) High-affinity binding through polyvalency, and 2) Steric stabilization.
Sigel et al. (J. Am. Chern. Soc., 118(16), 3789–3800, 1996) reported the efficacy of polymers containing sialoside groups in inhibiting the adhesion of influenza virus to, erythrocytes. They delineated the contributions of enhanced substrate ligand binding and steric considerations to efficiency of inhibition. These investigators reported sialic acid ligands, which can be exploited for the inhibition of the influenza virus. Monomeric inhibitor requires a higher concentration for inhibition since they are required to occupy at least half of the sialic acid binding sites on the virus, whereas the high molecular weight inhibitors need only a few attachments to achieve the same. Dimick et al. (J. Am. Chern. Soc., 121: 44, 10286, 1999) reported the molecular cluster glycoside effects, and the synthesis of polyvalent ligands for the plant lectin concanavalin A.
Krepinsky, et al. (U.S. Pat. No. 6,184,368, 2001) reported the limitations in the productive binding of chitosan to lysozyme and methods for the synthesis of polyvalent carbohydrate molecules by glycosylation of partially protected polysaccharides bearing a single glycosylating agent or a mixture of glycosylating agents.
Roseman D., S, et al., (J., Biol., Chem., 18;276 (20): 17052–7, 2001) reported greater specificity of mannose/N-acetylgalactosamine receptor for multivalent ligands than monovalent ligands. Various methods have been reported in the past to synthesize multivalent ligands such as ring-opening metathesis polymerization (ROMP). ROMP has been used to generate well defined, biologically active polymers by Gibson et al., (Chern. Comm., 1095–1096, 1997) and Biagini et al., (Polymer, 39, 1007–1014, 1998)
Damschroder et al. (U.S. Pat. No. 2,548,520, 1951) disclosed high molecular weight materials prepared by copolymerizing proteins conjugated with unsaturated monomers or proteins conjugated with preformed polymers. Synthesis of these high molecular weight materials generally requires temperatures up to 100 0 C. Such high temperatures are not well tolerated by most of the proteins. Thus the methods described are unsuitable for producing polymers of biologically active molecules.
Jaworek, et al. (U.S. Pat. No. 3,969,287, 1976) reports a method for the preparation of carrier-bound proteins, wherein the protein is reacted with a monomer containing at least one double bond capable of copolymerization. The carrier is provided as a water insoluble solid or is produced in situ by the polymerization of water-soluble monomers in the presence of the protein monomer conjugate. The proteins utilized in the method of this invention are typically enzymes.
The carbohydrate such as NAG serve as ligands for lectins and lysozyme. Roy et al. (J. Chem. Soc. Chem. Comm., 1611–1613, 1992) reported custom designed glycopolymer synthesis by terpolymerizations. The N-acryloyl precursors and the acrylamide were used as effector molecules to provide specific properties such as hydrophobicity and mimicking tyrosine residues of proteins.
Mochalova et ai. (Antiviral Research, 23, 179–190, 1994) reported carbohydrate inhibitors like sialic acid anchored to polymeric or liposomal carriers. They conjugated glycylamido benzylsialoside with poly(acrylic acid-co-acrylamides) and dextrans. These polymeric ligands were evaluated for their ability to bind influenza A and B virus strains in cell culture.
Hansen et al., (J. Am. Chem. Soc., 119, 6974, 1997) reported the ability to inhibit the binding of the bacterium Staphylococcus suis to Gal a (1,4) Gal on epithelial cells of the urinary tract. And the results represent the minimum concentration of multivalent compound required to inhibit the crosslinking of red blood cells by the bacterium.
Nishimura (Macromolecules 27, 4876–4880, 1994) demonstrated that the inhibitory effect of glycosylated-cyclodextrins on the erythrocytes agglutination induced by wheat germ (Triticum vulgaris) agglutinin was observed at 240-fold lower concentration that its monomeric counterpart.
In an alternative approach -Kanai, et aL (J. Am. Chern. Soc., 119, 9931–9932, 1997) reported ring opening metathesis polymerization (ROMP) methods for the synthesis. However the methods are complicated and do not control “living” nature of glycopolymer.
Dimick et al. (J. Am. Chern. Soc., 121, 44, 10286, 1999) explored newer strategies for enhancing interactions. Synthesis of polyvalent ligands was reported and the role of glycosidic clusters in enhancing binding with plant lectin concanavalin A was demonstrated.
Yamada et al (Macromolecules, 32, 3553–3558, 1999) reported controlled synthesis of amphiphilic block co polymers with pendant N-Acetyl Glucosamine (NAG) residues by living cationic polymerization. Copolymer architecture resulted in an enhancement in binding between Wheat Germ Agglutinin (WGA) and NAG.
Krepinsky, et al. (U.S. Pat. No. 6,184,368) reported methods for synthesis of polyvalent carbohydrate molecule by glycosylations of partially protected polysaccharides bearing a single glycosylating agent or a mixture of glycosylating agents. The patent explains the non-productive binding of chitosan to lysozyme.
Chitosan has the Formula 4) and is linear, binary heteropolysaccharide and consists of acetaamido-2-deoxy-β-D-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-D glucose (GlcNAc, D-unit). The active site of lysozyme comprises subsites designated A–F. Specific binding of chitosan sequences to lysozyme begins with binding of the NAG units in the subsite C. Moreover natural ligands derived from glucose are susceptible to microbial growth. There is need to synthesize ligands similar to repeat units of chitosan which will not be hydrolyzed by lysozyme. These polymers are expected to be more stable than chitin and chitosan. Apart from the type of the ligand, its distribution along the polymer chain also plays a crucial role in influencing the efficiency of the inhibition.
Natural oligosaccharides usually bind to their host molecules weakly and they must be used in larger quantities for an effective treatment. This problem can be overcome by synthesizing polyvalent carbohydrate conjugates (Zopf, D., Roth, S. Lancet 347, 1017, 1996) since such molecules bind the target molecules through multiple contacts and result in strong binding and also benefit from steric contribution arising out of their macromolecular nature.
Polyvalent molecules occupy the carbohydrate binding site competitively and the process of infection is thus interrupted. The polyvalent oligosaccharide molecule has a further advantage in that it can bind to particular pathogenic bacterium without any prior knowledge of exact binding requirements of the particular microorganism.
The design of multivalent ligands containing sialic acid is reported recently by Whitesides et al., (Angew. Chem. Int. Edn., 37, 2754–2794, 1998) for the treatment of infectious diseases such as rotavirus and influenza virus. This concept is justified on the basis of inhibition of virus adhesion to the host cells. The approach is therapeutically important because of increased pathogen resistance to antibiotics and drugs.
Oligosaccharide moieties, which bind to cellular proteins with higher specificity has a greater conformational flexibility. Various strategies have been suggested in the past to enhance the interactions.
Nishimora et al, (Tetrahedron 56, 9909, 2000) synthesized clustering, sugar homopolymers from Acrylamidoalkyl glycosides of N-Acetyl-D-Glucosamine. On addition of the cluster type polymer, binding to Wheat Germ Agglutinin was enhanced. Controlled synthesis of amphiphilic block copolymers bearing pend.ent N-Acetyl-D Glucosamine residues by living cationic polymerization and the interaction of the resulting diblock copolymers with lectins was reported by Yamada et al. (Macromolecules, 32, 3553–3558, 1999). This methodology of synthesizing homopolymers and the block copolymers with N-Acetyl-D-Glucosamine residues demonstrate significant increase in binding affinity for lectin.